Establishment of loop-mediated isothermal amplification method for detection of Legionella pneumophila / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish a simple and rapid molecular detection for Legionella pneumophila.</p><p><b>METHODS</b>The loop-mediated isothermal amplification (LAMP) was applied for detection of Legionella pneumophila. A set of primers were designed to identify six special areas in mip gene of Legionella pneumophila. Genomic DNAs from 13 bacterial strains,including 8 Legionella pneumophila strains and 5 other bacterial strains were amplified by LAMP and general PCR method to evaluate the specificity and sensibility of LAMP.</p><p><b>RESULT</b>All positive tubes produced visible white precipitation, and no precipitation was observed in others. By adding smart green fluorescent dye, all Legionella pneumophila positive tubes presented a strong green fluorescence, while others showed weak fluorescence. The detection rate of LAMP was higher than that of general PCR. The detection limits were 576fg with genomic DNA of Legionella pneumophila,and 8 cfu/mL with positive water samples.</p><p><b>CONCLUSION</b>LAMP detection of Legionella pneumophila is an effective and low-cost method with high specificity and sensitivity requiring no special equipment.</p>

Preparation and identification of a polyclonal antibody against novel cysteinyl leukotriene receptor GPR17 / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To prepare and identify a polyclonal antibody (pAb) against GPR17, a novel cysteinyl leukotriene receptor.</p><p><b>METHODS</b>Rabbits were immunized with KLH-coupled GPR17 peptide to prepare the pAb. The titer of the pAb in rabbit plasma was detected by indirect ELISA, and the specificity of the pAb was tested by antigen blockade. GPR17 tissue distribution was detected by Western blot with the pAb.</p><p><b>RESULTS</b>The pAb showed a titer as high as 1:16 364,and was not cross-reacted with the antigens of CysLT(1) and CysLT(2) receptors. A higher expression of GPR17 in the rat brain and heart was detected using the newly prepared pAb. The molecular weigh of GPR17 protein was about 43 kD.</p><p><b>CONCLUSION</b>The prepared GPR17 pAb has high sensitivity and specificity,and can be used in Western blot for detecting GPR17.</p>

Preparation and identification of polyclonal antibody against cysteinyl leukotriene receptor 2 / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To prepare and identify a polyclonal antibody against cysteinyl leukotriene receptor (CysLT(2)receptor).</p><p><b>METHODS</b>Rabbits were immunized with KLH-coupled CysLT(2) receptor peptide to prepare the polyclonal antibody (pAb). The titer of the pAb in rabbit plasma was detected by indirect ELISA, and the specificity of the pAb was tested by antigen blockade. The tissue distribution of CysLT(2) receptor was detected by Western blot and immunohistochemistry with the prepared pAb.</p><p><b>RESULT</b>The pAb showed a titer higher than 1/1047296, and was specific to CysLT(2) receptor, without cross-reaction with the antigens of CysLT(1) receptor and GPR17. A higher expression of CysLT(2) receptor in kidney, brain and lung of rats and mice was detected by Western blot analysis using the prepared pAb. The molecular weight of CysLT(2) receptor protein was about 40 kD. Immunohistochemical examination showed that CysLT(2) receptor was expressed mainly in the neuron, and partly in astrocytes in rat brain.</p><p><b>CONCLUSION</b>The prepared CysLT(2) receptor pAb has high sensitivity and specificity, and can be used in Western blot and immunohistochemistry.</p>

Method for screening cysteinyl leukotriene receptor 2 antagonists and preliminary screening of compounds / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish a method for screening cysteinyl leukotriene receptor 2 (CysLT(2)) antagonists and to preliminarily screen a series of synthetic compounds.</p><p><b>METHODS</b>Rat glioma cell line (C6 cells) highly expressing CysLT(2) receptor was used. Intracellular calcium concentration was measured after stimulation with the agonist LTD(4),which was used to screen compounds with antagonist activity for CysLT(2) receptor. Bay u9773, a CysLT1/CysLT(2) receptor non-selective antagonist, and AP-100984, a CysLT(2) receptor antagonist, were used as control.</p><p><b>RESULT</b>PT-PCR showed a higher expression of CysLT(2) receptor in C6 cells. LTD(4) at 1 mumol/L significantly increased intracellular calcium in C6 cells; the maximal effect was about 37.5% of ATP, a positive stimulus.LTD(4)-induced increase of intracellular calcium was blocked by CysLT(2) receptor antagonists, but not by CysLT(1) receptor antagonists. Among the synthetic compounds, D(XW-)1,2,13,23,29 and 30 inhibited LTD(4)-induced increase of intracellular calcium.</p><p><b>CONCLUSION</b>LTD(4)-induced change in intracellular calcium in C6 cells can be used as a screening method for CysLT(2) receptor antagonists. The compounds, D(XW-)1,2,13,23,29 and 30, possess antagonist activity for CysLT(2) receptor.</p>

Adhesional inhibiton of polyclonal anti-porin I antibody / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the blockness effects of purified polyclonal anti-porin I antibody on N. gonorrhoeae adherence to genitourinary tract epithelia of BALB/c mouse.</p><p><b>METHODS</b>Polyclonal anti GST-PI antibody was generated by immunizing rabbit with GST-PI fusion protein which was constructed and expressed by ourselves. The purified immunoglobulin G was obtained by ammonium sulphate deposition and DEAE cellulose chromatography. Mice model of gonorrhea was established. In order to evaluate the effects of PI-IgG on gonococcus adhesion to vagina mucus, the macroscopic and pathological assessing as well as gonococcus culture was employed after gonococcus challenge on PI-IgG immunized mice.</p><p><b>RESULT</b>No pus and pathological inflammation were observed on mice vagina mucus treated with 1 mg/ml PI-IgG 3 hours before gonococcus challenge. Gonococcus could not be detected in the smears and washing solutions from vagina. Pathological inflammation was found in mice treated with anti PI-IgG, in which the concentrations were lower than 1 mg/ml or the treated time was longer than 3 hours prior to gonococcus challenge.</p><p><b>CONCLUSION</b>The purified anti PI-IgG can effectively inhibit the adherence and infection of gonococci to genitourinary tract epithelia of BALB/c mice. In addition, the blocking duration of anti PI-IgG is associated with antibody concentration.</p>

Expression of tyrosine phosphatase containing C-src homology SH-2 in benign prostate hyperplasia / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To explore the expression of tyrosine phosphatase containing C-src homology SH-2 (SHP-1 and SHP-2) in benign prostate hyperplasia.</p><p><b>METHODS</b>With En Vision two-step method, the expression of SHP-1 and SHP-2 was detected in 10 cases of normal prostate tissue, 30 cases of BPH, 20 cases of PIN, 20 cases of high differential Pca and 20 cases of low differential Pca.</p><p><b>RESULT</b>The expression of SHP-2 in normal group was mainly distributed in the cytoplasm of secretive cells and basal cells, and a little part in the nucleu. In BPH it was distributed equally in the plasm and nucleu. In PIN, high differential Pca and low differential Pca, SHP-2 expressed mainly in nucleu. The average dyeing index of SHP-2 in each group is 0.4, 1.7, 2.1, 2.2 and 2.6. SHP-1 positive expression in normal prostate, BPH, PIN and high differential Pca showed differentiating layer staining in the cytoplasm of secretive cells and basal cells, while not in low differential Pca. The average dyeing index of SHP-1 in each group is 1.8, 1.8, 1.5, 1.2 and 0.4.</p><p><b>CONCLUSION</b>There are transformation in signal transduction relation with SHP-1 and SHP-2 in the progress of prostate cell proliferation, differentiation and malignant. The abnormal activation and distribution of SHP-2 might induce prostate reconstruction and hyperplasia, even carcinoma.</p>

Experiment treatment of collagen-induced arthritis in rats with recombinant plasmid containing vasoactive intestinal peptide gene / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the therapeutical effect of recombinant plasmid containing vasoactive intestinal peptide gene (pcDNA3.1+/VIP) on collagen-induced arthritis (CIA) in rats.</p><p><b>METHODS</b>The experimental arthritis was induced by intradermal injection of bovine type II collagen emulsified in Freund's adjuvants in male SD rats. The rats then were given intra-articular injection with recombinant plasmid (pcDNA3.1+/VIP). The levels of serum TNF-alpha, IL-4 and IL-2 were detected by Avidin-Biotin Peroxdase Complex-enzyme-linked immunosorbent assay (ABC-ELISA) and the pathological changes in the joint of rats were observed.</p><p><b>RESULT</b>Histological examination showed massive inflammatory infiltration in the joint with destruction of bone and cartilage, while the severity of pathological changes in synovia of VIP-treated rats was markedly reduced. Compared with normal group, the serum TNF-alpha, IL-2 levels of CIA rats were significantly increased (P <0.05) and IL-4 level was decreased (P<0.05). Compared with control and pcDNA3.1+ -treated CIA rats, serum TNF-alpha and IL-2 levels of pcDNA3.1+/VIP-treated rats were decreased and IL-4 level was increased (P<0.05).</p><p><b>CONCLUSION</b>Recombinant plasmid containing vasoactive intestinal peptide gene (pcDNA3.1+/VIP) can reduce the clinical and histological severity of established CIA and it might be a promising candidate for treatment of rheumatoid arthritis.</p>

Preparation of monoclonal antibodies against human mesenchymal stem cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To prepare monoclonal antibodies (McAbs) against human mesenchymal stem cells (hMSCs) and to study their biological characteristics.</p><p><b>METHODS</b>BALB/C mice were immunized with pooled hMSCs. McAbs were prepared by hybridoma technique and their biological characteristics were analyzed by indirect immunofluorescence, immunohistochemistry and flow cytometry.</p><p><b>RESULT</b>Five hybridoma cell lines were successfully established, which secret McAbs specifically against hMSCs. Investigations showed that all these McAb reacted only to hMSCs and had no cross-reaction to other human cells, the relative affinities of 5 McAbs were 1x10(6) (ZUB1), 1x10(5) (ZUB4), 1x10(6) (ZUC3), ZUE12 (1x10(5)) and 1x10(5) (ZUF10), respectively. Isotype analysis showed that ZUB1, ZUE12, ZUF10 against the same isotype, while ZUC3, ZUB4 against other two different isotypes alone. Flow cytometric analysis showed that the positive expression rate of cultured hMSCs was 87.39% (ZUB1), 88.07% (ZUB4), 88.12% (ZUC3), 69.89% (ZUE12) and 83.67% (ZUF10).</p><p><b>CONCLUSION</b>The prepared five McAbs can specifically react against hMSCs, which can be used for selection and study of hMCSs.</p>

Study on lysosomes degradation of ricin A chain / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To study lysosomes involvement in the degradation of ricin A chain.</p><p><b>METHODS</b>A lysosome-targeted singal KFERQ was added to the C terminus of rRTA by DNA recombinant technology. A pKK223.3 expression system in E. coli was used to produce recombinant ricine A chain (rRTA) and rRTA-KFERQ. Recombinant proteins were purified by affinity chromatography using Blue-Sepharose 6B. The cytotoxicity of recombinant proteins was measured by the MTT method.</p><p><b>RESULTS</b>Recombinant RTA-KFERQ was 49.87%, 54.18% and 88.68% less cytotoxic than RTA itself on the three cell lines HEPG2, Hela and A549, respectively.</p><p><b>CONCLUSION</b>Lysosomes can degrade, but not completely inactivate RTA in different cells, suggesting cells may have other degradation pathways for RTA.</p>

Therapeutic effect of cationic liposome-mediated interleukin-12 gene delivery on murine melanoma in vivo / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the therapeutic effect of cationic liposome-mediated interleukin-12 gene delivery on established murine melanoma in vivo.</p><p><b>METHODS</b>The lipofectin encapsulated pCmIL-12 plasmid was given to C57BL/6 mice on the day 3,5,7,9 after inoculation of B16 melanoma cells. The tumor size, the survival time of mice and the NK cell activity were observed.</p><p><b>RESULTS</b>The pCmIL-12 plasmid coupled with cationic liposome inhibited the tumor growth and improved the survival of mice bearing established melanoma. The activity of NK cells was also enhanced after interleukin-12 gene delivery in vivo.</p><p><b>CONCLUSION</b>Cationic liposome-mediated interleukin-12 gene delivery has significantly therapeutic effects on mice melanoma in vivo.</p>

Construction of recombinant plasmid containing mouse vasoactive intestinal peptide gene and its expression in COS-7 cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct the eukaryotic expression plasmid containing mouse vasoactive intestinal peptide(VIP) gene with biological activities.</p><p><b>METHODS</b>VIP cDNA including the sequences of signal peptide was cloned from mouse thymus by RT-PCR, and then inserted into the mammalian expression vector pcDNA3.1 between Hind III and EcoR I restriction sites. COS-7 cells were transfected with pcDNA3. 1-VIP using liposome, the expression of VIP was identified by Western blot and ELISA. Supernatant of transfected cell culture was added to LPS-stimulated macrophages and the TNF-alpha production in cell medium was observed by ELISA.</p><p><b>RESULTS</b>The cloned VIP cDNA was confirmed by enzyme digestion and DNA sequencing. The expression of VIP was detected in the pcDNA3. 1-VIP transfected COS-7 cells by Western blot and ELISA. The VIP in culture supernatant potently inhibited TNF-alpha production by LPS-induced Macrophages in vitro.</p><p><b>CONCLUSION</b>The eukaryotic expression plasmid that expresses biological active murine VIP has been constructed successfully.</p>

Expression of GST-HAI-1 fusion protein and development of monoclonal antibody against human hepatocyte growth factor activator inhibitor 1 / 生物工程学报

The aim of this study is to develop monoclonal antibody against human hepatocyte growth factor activator inhibitor 1 (HAI-1) for future study of HAI-1. The cDNA fragments of human hepatocyte growth factor activator inhibitor 1 (HAI-1) were subcloned to construct GST-HAI-1 fusion protein expression vectors. The vectors were transformed into E. coli and fusion protein expression was induced by IPTG. The GST-HAI-1 fusion proteins were separated on preparative SDS-PAGE and recovered by electroelution, and used to immunize BALB/c mice. Hybridomas producing monoclonal antibodies against human HAI-1 were prepared by cell fusion technique and characterized by ELISA, Western Blot and immunohistochemical staining. One hybridoma cell line, ZMC6, was obtained, which produces specific antibody against the expressed GST-HAI-1 fusion protein. The monoclonal antibody recognizes both the membrane-type and secretory-type HAI-1 proteins of colorectal tissue. The successful development of anti-HAI-1 antibody provides a powerful tool for further investigation on HAI-1's function.

Preparation of monoclonal antibodies against oh(8)dG and their biological characteristics / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To prepare monoclonal antibodies against oh(8)dG and to evaluate the relationship between Hp infection and oxidative DNA damage by detecting oh8dG in gastric mucosa.</p><p><b>METHODS</b>BALB/C mice were immunized with BSA-oh(8)dG conjugate, monoclonal antibodies were prepared by hybridoma technique, the biological characteristics of antibodies were analysed by competitive ELISA, Western blot and immunohistochemistry.</p><p><b>RESULTS</b>Two strains of hybridoma cell were obtained. ELISA and Western blot indicated that the antibodies were fairly specific for oh(8)dG. In immunohistochemistry,the positive rate of oh(8)dG expression in Hp positive tissues and Hp negative tissues was 55% and 5%, respectively(P<0.01).</p><p><b>CONCLUSION</b>The prepared antibodies can specially recognize oh(8)dG and immunohistochemistry with the monoclonal antibodies showed Hp infection can increase oh(8)dG level in gastric mucosa.</p>

Construction of bi-cistronic co-expression plasmid of mIL-12 and the expression in vitro or in vivo / 浙江大学学报·医学版

OBJECTIVE: To construct a bi-cistronic co-expression plasmid for mouse interleukin-12 and to observe its expression in vitro or in vivo.METHODS: The full-length cDNA encoding p35 and p40 was cloned into eukaryotic cells expression vector pcDNA 3.1 respectively. Subsequently,the p35 expression unit was inserted into pcDNA 3.1/p40 to produce the bi-cistronic co-expression plasmid in which the p35 and p40 genes were controlled by their own CMV.The plasmid was expressed in vitro and in vivo. RESULTS: The mIL-12 in the supernatant was detected by ELISA after the pCmIL-12 was transfected into COS-7 cells. The activity of NK cells could be augmented by the supernatant in vitro and also by by intradermal delivery of pCmIL-12 in vivo. CONCLUSION: The plasmid constructed by us can express biologically active mIL-12 in vitro and in vivo.